Project Abstract Diversity-generating retroelements (DGRs) are molecular evolution machines found in bacteria, archaea and their viruses. They diversify protein-encoding sequences to facilitate the adaptation of their hosts to changing environments. Hypervariation results from an error-prone retrotransposition process called mutagenic homing, which transfers sequence information from a template repeat (TR) to a variable repeat (VR) that results in adenine to random nucleotide conversions. The long-term goal of the PI?s group is to understand the mechanism of DGR mutagenic homing and to develop them for practical applications. In analogy to related retroelements, DGR homing was proposed to occur through a target DNA-primed reverse transcription mechanism. Interestingly, recent discoveries by the PI?s group showed that reverse transcription of TR of the Bordetella phage DGR is primed by a downstream adenine residue of the RNA intermediate and is target (VR)-independent. Intriguingly, the TR RNA intermediate was found to be nicked in a bRT (Bordetella reverse transcriptase)-dependent manner to generate a 3?-OH for cDNA priming, and single amino acid substitutions at the RT catalytic core abolish the nicking activity, suggesting that bRT plays a catalytic role in the cleavage reaction. Adenine-specific mutagenesis occurs during (?)cDNA synthesis and results from misincorporation of standard deoxyribonulceotides by bRT. In addition, mutational analysis showed that this special, target-independent reverse transcription reaction is responsible for DGR mutagenic homing, revealing a novel mechanism of DNA hypervariation. The specific aims are based on these novel discoveries. Aim 1 will characterize the secondary structure of TR RNA and map the Avd and bRT binding sites on the RNA intermediate. These studies will generate the first secondary structure of a DGR RNA, and reveal whether the TR RNA intermediate is catalytically cleaved by bRT. Aim 2 will determine the mechanism of cDNA integration at the 3? and 5? ends of VR. Roles of base pairing interactions between the RNA primer and VR DNA and between cDNA and VR DNA in 3? cDNA integration will be tested. These studies may lead to discovery of novel cDNA integration mechanisms. Aim 3 will determine the mechanism of adenine-specific mutagenesis, which is a hallmark of DGRs. Understanding the mechanism of adenine-specific mutagenesis will likely yield new insights on RT fidelity issues. In summary, studies proposed in this application will elucidate the mechanism of DGR mutagenic homing, which may have broad implications in health and science.